Universidade de São Paulo

2016

Micro-computed tomography scan and virtual

histological slide data for the land planarian

Obama otavioi (Platyhelminthes) GigaScience. 2016 Mar 16;5(1):13http://www.producao.usp.br/handle/BDPI/49956

Downloaded from: Biblioteca Digital da Produção Intelectual - BDPI, Universidade de São Paulo

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Micro-computed tomography scan and virtualhistological slide data for the land planarianObama otavioi (Platyhelminthes)Carbayo and Lenihan

Carbayo and Lenihan GigaScience (2016) 5:13 DOI 10.1186/s13742-016-0119-4

DATA NOTE Open Access

Micro-computed tomography scan andvirtual histological slide data for the landplanarian Obama otavioi (Platyhelminthes)Fernando Carbayo1* and Jennifer Winifred Lenihan2

Abstract

Background: We investigated whether images obtained through X-ray micro-computed tomography (μCT) can be usedin conjunction with traditional methods for morphological studies of soft-bodied land planarians. μCT is non-invasive andprovides true-to-scale three-dimensional imagery at high resolution. We compared μCT-based images of a recentlydescribed land planarian species of Obama otavioi (Platyhelminthes) with those obtained from light microphotography ofhistological sections, most of which were also digitized at high magnification.

Findings: The specimens studied were collected in 2012. Subsequent μCT-based images of the stained body of aparatype show nearly all morphological features provided by traditional histology, with the exception of particularlyminute structures, smaller than 5 μm, such as the sensory pits and single muscle fibers, which are best visible ontraditional histological sections. Because the technique is non-destructive, the scanned specimen is preserved withoutdamage. The raw and derivative μCT data and virtual histological sections are freely available in GigaDB.

Conclusions: The μCT datasets of these stained soft-bodied organisms reveal images of external and internal structuresthat support previous taxonomic studies. This technique can be particularly important for non-destructively revealinginternal details of whole museum specimens at a faster rate than histology alone. High-resolution virtual histological slidesalso allow further searches for new, previously unstudied morphological features. The use of X-ray equipment with higherresolution can enable smaller sensory organ and muscle fiber details to be seen. The image sets, μCT-based images anddigitized histological slides can be disseminated without the constraints of specimen loans.

Keywords: μCT, MicroCT, Slide digitization, Anatomy, Histology, Taxonomy, Morphology, Geoplanidae, Tricladida, Soft-tissue

Data descriptionPurpose of data acquisitionUnequivocal identification and comprehensive descriptionof a diversity of animal taxa depend on destructivetechniques, such as traditional histological sections. Thistechnique is laborious and may result in patchy and dis-torted sections [1, 2]. Current taxonomic studies of landplanarians (Platyhelminthes, Tricladida) require histologicalsectioning. Furthermore, some land planarian species of thegenus Obama (Geoplaninae, a group of exclusively neo-tropical land planarian species) are morphologically very

similar to their relatives and taxonomically relevant organs,such as the copulatory apparatuses, are internally asymmet-rical so histological sections often hinder reconstruction ofthese structures [3].Many species of Geoplaninae are poorly represented in

museum collections. Destructive techniques provide mor-phological information along with a sliced, chemicallymodified tissue that cannot be used for subsequent ap-proaches, such as DNA analysis. Thus, whenever available,type specimens are best candidates to be studied throughnon-destructive techniques, such as X-ray micro-computedtomography (μCT). Here we present μCT scans of a para-type of the recently described species of land planarianObama otavioi Carbayo, 2016 [4] along with virtual histo-logical sections of the holotype and another paratype of thespecies. The whole type-material on which description of

* Correspondence: baz@usp.br1Laboratório de Ecologia e Evolução, Escola de Artes, Ciências eHumanidades, Universidade de São Paulo - USP, Av. Arlindo Bettio, 1000, CEP03828-000 São Paulo, SP, BrazilFull list of author information is available at the end of the article

© 2016 Carbayo and Lenihan. Open Access This article is distributed under the terms of the Creative Commons Attribution4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Carbayo and Lenihan GigaScience (2016) 5:13 DOI 10.1186/s13742-016-0119-4

the species was based consists of only these threespecimens.

Studied specimensThree specimens comprise the type series. They werecollected in Reserva Biológica do Alto da Serra deParanapiacaba, Santo André, State of São Paulo, Brazil(-23.77697, -46.31212), on 22 September 2012. Photo-graphs of the living animals were taken in the fieldand in the laboratory with Sony DSC-W125, SonyDSC-W310 and Canon EOS Rebel T5i digital camerasand are freely available at [5]. They were fixed in 10 %formalin and preserved in 80 % ethanol, with the ex-ception of a small piece of tissue that before fixationwas cut off and frozen in absolute ethanol.

Data acquisition and processingMicro-computed tomography procedureThe paratype MCZ 59100 was submerged in a solution of0.3 % phosphotungstic acid (PTA) and 3 % dimethyl sulfox-ide (DMSO) in 95 % hydrated ethanol, following the proto-col by Fernández et al. [6] for earthworms. The time ofimmersion in this solution was extended to 90 days. Thespecimen was then rinsed in 95 % ethanol and placed intoa plastic straw with 95 % ethanol for preliminary scans.Scans were performed with the X-ray μCT SkyScan 1173scanner (Bruker MicroCT, Kontich, Belgium) equippedwith a Hamamatsu 130/300 tungsten X-ray source and aFlat Panel Sensor camera detector with 2,240 × 2,240 pixels.To enhance the contrast, the specimen was restained with1 % iodine in absolute ethanol (see detailed description inCarbayo et al. [4]) and new scans were performed. The finaldatasets obtained consist of four scans (Table 1, Fig. 1a),one at low resolution of the entire body and three scans, athigher resolution, of the anterior tip of the body, the phar-ynx and the copulatory apparatus, respectively. The scan ofthe entire specimen at low resolution elucidated theorganization and distribution of internal organs, whereasthe remaining scans, taken at higher resolution, were aimedat viewing minute structures. Scanning parameters for thefour scans are summarized in Table 1; additional parame-ters can be found in the log file (.log) of each dataset folderavailable at GigaDB [7]. Each scan resulted in a set of pro-jection images in tagged image file format (TIFF, .tif). Nobinning protocols were used during data acquisition. Theprojection images covered 2,240 × 2,240 pixels at 16-bit dy-namic range. Reconstruction of the two-dimensional (2D)projection images into a three-dimensional (3D) volumetricimage stack was performed using the software NRecon1.6.6.0 (Bruker microCT, Kontich, Belgium). This programruns under the reconstruction engine NReconServer 1.6.6.The output format for the 3D volumetric image stacks wasbitmap image file (BMP,.bmp) at 8-bit dynamic range and2,240 × 2,240 pixel size, but they can also be reconstructed

as a TIFF. In order to reduce final file size, the volume ofinterest) function, a 3D cropping tool, was used to removeuninformative parts of the data following reconstruction.This resulted in changes to the overall pixel dimensions ofeach reconstructed image stack but did not lead to spatialdistortions in any of the three dimensions.

Histological slide digitizationThe holotype and paratype MZUSP PL 1573 were histolog-ically processed, sectioned and stained using the Mallorymethod as modified by Cason [8]. A total of 166 histo-logical slides were obtained from the holotype (Fig. 1b), and107 from paratype MZUSP PL 1573, as follows: HolotypeMZUSP PL 1574 (field number, F5470): anterior end: trans-verse sections on 40 slides; anterior region 2: sagittal sec-tions on 35 slides; anterior region 3: horizontal sections on24 slides; pharynx: sagittal sections on 28 slides; copulatoryapparatus: sagittal sections on 49 slides. Paratype MZUSPPL 1573 (field number, F5435): pharynx: sagittal sectionson 46 slides; copulatory apparatus: sagittal sections on 61slides (Fig. 1c).A total of 81 histological slides were digitized with slide

scanner Zeiss Axio Scan Z1 (Table 2). For each slide, apreview was taken for automatic tissue detection. Theseselections were then refined manually. To obtain high-resolution images, tissue sections were subdivided intomultiple small images, or tiles, that were photographedwith bright field imaging mode with a 40× objective.Focus was applied every fourth or sixth tile. Z-stackingwas activated to obtain a single sharp plane image; this isvaluable when sections on the histological slides are un-even. ZEN software (Carl Zeiss Microscopy) automaticallyproduced aligned tiles as a single virtual histological slidein.czi format, which are freely available at GigaDB [7].

Data qualityμCT-based imagesHighest contrast and sharpness were achieved at about34–35 kV, 160–190 μA and 1,000–1,200 ms exposure.The highest voxel resolution achieved with the scan was6.04 μm. High quality scans of the entire animal wereavoided as to not create large file sizes. Instead, eachindividual was scanned at lower resolution and smallsections were scanned at high resolution to generatemore manageable file sizes.Eyes and the prostatic vesicle are small structures that

are less easily outlined in the scans. Eyes were partiallymasked by body pigmented cells. Despite its relativelylarge size, the vesicle stained weakly so it was laboriousto outline it. Structures less than 5 μm in size, such assensory pits and single muscle cells, were also nearly in-distinguishable on the μCT-based images.

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Virtual histological slidesAlmost all images of virtual histological slides obtainedwere of high quality. A few images present blurry por-tions or border tiles were visible. This might be due totiny debris remaining on the cover slides and to tech-nical failure at digitization, respectively. Given that theseregions occurred very scattered along the serial sections,

they do not hinder image analyses aimed at taxonomicinvestigation.

Potential usesFreely available digital image datasets are suitable forstudy of museum specimens across the globe, withoutthe potentially prohibitive costs of travel or museum

Fig. 1 Type-specimens of Obama otavioi studied. a Ventral view of a 3D rendering of the μCT dataset of paratype MCZ 59100. Dashed linesindicate portions of the body scanned separately: anterior end, pharynx and copulatory apparatus. b Outline of holotype with indication ofportions of the body that were histologically sectioned. c Outline of paratype MZUSP PL 1573 and portions of the body that werehistologically sectioned

Table 1 Overview of the paratype MCZ 59100 mCT dataset deposited in GigaDB

Region of the body Entire body (except tail) Anterior end Pharynx Copulatory apparatus

Image pixel size 9.95 6.04 6.04 6.04

Source voltage (kV) 35 34 35 35

Source current (μA) 170 160 160 190

Exposure (msec) 1050 1200 1200 1200

Degree rotation step 0.16 0.07 0.07 0.1

Frame averaging 6 9 12 14

Connected scans 5 3 1 1

Projection files 7505 x.tif, 5 x.log 7202 x.tif, 3 x.log 2401 x.tif, 1 x.log 2401x.tif, 1 x.log

Name of projection files 28-F5434-inteiro19abril_#.tif 27-F5434-PA_#.tif 28-F5434-F_18abril_#.tif F5434-otavioi-AC_VI_#.tif

Total size of projectionfiles (GB)

64 72.3 24.1 24.1

Reconstruction files 7241 x.bmp 1 x.log 4353 x.bmp, 1 x.log 1903 x.bmp, 1 x.log 1832 x.bmp, 1 x.log, 1 x.db, 2x.tif

Name of reconstruction files 28-F5434-inteiro19abril__rec#.bmp

27-F5434-PA__rec#.bmp

28-F5434-F_18abril__rec#.bmp

F5434-otavioi-AC_VI__rec#.bmp

Total size of reconstruction files(GB)

7 5.5 4.7 4.7

See also Fig. 1a

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Table 2 Overview of the virtual slides of the holotype and paratype MZUSP PL 1173 dataset deposited in GigaDB

Specimen Holotype Paratype MZUSP PL 1573

Region ofthe body

Anterior end Ovaries Behind ovaries Pharynx Copulatoryapparatus

Pharynx Copulatory apparatus

Name of filefolder

09-Holotype(MZUSPPL1574)-histology-AntEnd

10-Holotype(MZUSPPL1574)-histology-Ovaries

11-Holotype(MZUSPPL1574)-histology-behindOvaries

12-Holotype(MZUSPPL1574)-histology-Pharynx

13-Holotype-Histology-CopApp

14-Paratype-MZUSP-PL1573(F5435)-histology-Pharynx

15-Paratype-MZUSP-PL1573(F5435)-histology-Copulatory

Plane ofsection

Transverse Sagittal Horizontal Sagittal Sagittal Sagittal Sagittal

Slidesdigitized (oftotal)

10 (40) 9 (35) 11 (24) 8 (28) 15 (49) 10 (46) 18 (61)

Total size ofscans (GB)

6.96 28.2 47.6 32.5 73.7 44.2 57.7

See also Fig. 1b,c

Carbayo

andLenihan

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loans. These cybertypes [9] can be non-destructivelystudied in ways that would otherwise damage tissues orprevent DNA extraction. The non-destructive nature ofμCT allows for the study of type specimens, and for mo-lecular work after scanning. Furthermore, μCT lends it-self to rapid mass scanning of taxonomic groups. Thismethod of visualization could then be implemented todistinguish minute morphological characters and pos-sibly identify new structures for taxonomic divisionamong cryptic species. Virtual slides allow verification ofmanual 3D reconstructions, which in part depend on in-dividual interpretation of the serial sections (see examplein [10]). They contain much more information thanwhat is described in taxonomic approaches, so they alsoallow the exploration of new unstudied morphologicalcharacters.

DiscussionAlthough these animals are relatively large, some of thetaxonomically relevant structures, such as sensory pits andmuscle organization, require high resolution of μCT-basedimages to be detected. Consequently, high-performancecomputers with suitable memory RAM and hard disk spaceare needed, and a rapid internet connection is essential.With these technological requirements, μCT-based imageryand virtual histological slides will be the best way to sur-mount the hindrance of specimen loans. μCT is especiallyrelevant for getting access non-destructively to morphologyof rare or unique specimens, such as type specimens.Digitization of histological slides of type specimens is thebest way to facilitate study of this material without the in-herent risk of damage when transporting loans.

Availability and requirementsData availabilityThe datasets are available at GigaDB [7]. They consist offour folders containing μCT-based images plus sevenfolders with virtual histological slides. Dataset name:μCT scans and digitized histological slides of type seriesof the land planarian Obama otavioi.

Data requirementsDownloaded reconstructed μCT-based images can be vi-sualized using the ‘File:Import: Image Sequence’ com-mand chain in the Java-based imaging software ImageJ[11]. There is also other 2D and 3D free visualizationsoftware [12]. Datasets can also be opened and manipu-lated using the freely available software from SkyScan inboth 2D and 3D formats [13]. For μCT-based images acomputer system with over about 24 GB random accessmemory (RAM) should be used.Downloaded virtual histological sections can be visual-

ized using Bio-Formats Importer plugin installed in theFiji distribution of ImageJ [14]. They also can be visualized

with ZEN. For virtual slides a computer system with overabout 4 GB main RAM should be used.

AbbreviationsμCT: micro-computed tomography.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsConceived and designed the experiments: FC. Performed the experiments:FC JL. Analyzed the data: FC JL. Contributed reagents/materials/analysis tools:FC JL. Wrote the paper: FC JL. Both authors read and approved the finalmanuscript.

AcknowledgementsInstituto de Botânica da Secretaria do Meio Ambiente do Estado de SãoPaulo licensed the fieldwork. Marcos Santos Silva, Ana Laura Almeida andAmanda Cseh helped with sampling. Ana Cristina Vasconcellos madehistological sections. José Ernesto Belizario and Beatriz Viana dos Santosprovided support with obtaining virtual histological slides (USP Proc.2012.1.17660.1.0). Beka Buckman kindly read the manuscript for Englishimprovement. São Paulo Research Foundation (FAPESP, Proc. 2014/13661-8)provided financial support to FC. This work was also supported by theMuseum of Comparative Zoology, Harvard University.

Author details1Laboratório de Ecologia e Evolução, Escola de Artes, Ciências eHumanidades, Universidade de São Paulo - USP, Av. Arlindo Bettio, 1000, CEP03828-000 São Paulo, SP, Brazil. 2Museum of Comparative Zoology,Department of Invertebrate Zoology, Harvard University, Cambridge,Massachusetts, USA.

Received: 2 December 2015 Accepted: 5 March 2016

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